Plant pathogenic bacterium Ralstonia solanacearum can rapidly evolve tolerance to antimicrobials produced by Pseudomonas biocontrol bacteria.

Soil-borne plant pathogens significantly threaten crop production due to lack of effective control methods. One alternative to traditional agrochemicals is microbial biocontrol, where pathogen growth is suppressed by naturally occurring bacteria that produce antimicrobial chemicals. However, it is still unclear if pathogenic bacteria can evolve tolerance to biocontrol antimicrobials and if this could constrain the long-term efficacy of biocontrol strategies. Here we used an in vitro experimental evolution approach to investigate if the phytopathogenic Ralstonia solanacearum bacterium, which causes bacterial wilt disease, can evolve tolerance to antimicrobials produced by Pseudomonas bacteria. We further asked if tolerance was specific to pairs of R. solanacearum and Pseudomonas strains and certain antimicrobial compounds produced by Pseudomonas. We found that while all R. solanacearum strains could initially be inhibited by Pseudomonas strains, this inhibition decreased following successive subculturing with or without Pseudomonas supernatants. Using separate tolerance assays, we show that the majority of R. solanacearum strains evolved increased tolerance to multiple Pseudomonas strains. Mechanistically, evolved tolerance was most likely linked to reduced susceptibility to orfamide lipopeptide antimicrobials secreted by Pseudomonas strains in our experimental conditions. Some levels of tolerance also evolved in the control treatments, which was likely correlated response due to adaptations to the culture media. Together, these results suggest that plant-pathogenic bacteria can rapidly evolve increased tolerance to bacterial antimicrobial compounds, which could reduce the long-term efficacy of microbial biocontrol.

Combining in vitro and in vivo screening to identify efficient Pseudomonas biocontrol strains against the phytopathogenic bacterium Ralstonia solanacearum.

Although plant pathogens are traditionally controlled using synthetic agrochemicals, the availability of commercial bactericides is still limited. One potential control strategy could be the use of plant growth-promoting bacteria (PGPB) to suppress pathogens via resource competition or the production of antimicrobial compounds. This study aimed to conduct in vitro and in vivo screening of eight Pseudomonas strains against Ralstonia solanacearum (the causative agent of bacterial wilt) and to investigate underlying mechanisms of potential pathogen suppression. We found that inhibitory effects were Pseudomonas strain-specific, with strain CHA0 showing the highest pathogen suppression. Genomic screening identified 2,4-diacetylphloroglucinol, pyoluteorin, and orfamides A and B secondary metabolite clusters in the genomes of the most inhibitory strains, which were investigated further. Although all these compounds suppressed R. solanacearum growth, only orfamide A was produced in the growth media based on mass spectrometry. Moreover, orfamide variants extracted from Pseudomonas cultures showed high pathogen suppression. Using the "Micro-Tom" tomato cultivar, it was found that CHA0 could reduce bacterial wilt disease incidence with one of the two tested pathogen strains. Together, these findings suggest that a better understanding of Pseudomonas-Ralstonia interactions in the rhizosphere is required to successfully translate in vitro findings into agricultural applications.

Molecular characterization of Pseudomonas from Agaricus bisporus caps reveal novel blotch pathogens in Western Europe.

BACKGROUND: Bacterial blotch is a group of economically important diseases affecting the cultivation of common button mushroom, Agaricus bisporus. Despite being studied for more than a century, the identity and nomenclature of blotch-causing Pseudomonas species is still unclear. This study aims to molecularly characterize the phylogenetic and phenotypic diversity of blotch pathogens in Western Europe. METHODS: In this study, blotched mushrooms were sampled from farms across the Netherlands, United Kingdom and Belgium. Bacteria were isolated from symptomatic cap tissue and tested in pathogenicity assays on fresh caps and in pots. Whole genome sequences of pathogenic and non-pathogenic isolates were used to establish phylogeny via multi-locus sequence alignment (MLSA), average nucleotide identity (ANI) and in-silico DNA:DNA hybridization (DDH) analyses. RESULTS: The known pathogens "Pseudomonas gingeri", P. tolaasii, "P. reactans" and P. costantinii were recovered from blotched mushroom caps. Seven novel pathogens were also identified, namely, P. yamanorum, P. edaphica, P. salomonii and strains that clustered with Pseudomonas sp. NC02 in one genomic species, and three non-pseudomonads, i.e. Serratia liquefaciens, S. proteamaculans and a Pantoea sp. Insights on the pathogenicity and symptom severity of these blotch pathogens were also generated. CONCLUSION: A detailed overview of genetic and regional diversity and the virulence of blotch pathogens in Western Europe, was obtained via the phylogenetic and phenotypic analyses. This information has implications in the study of symptomatic disease expression, development of diagnostic tools and design of localized strategies for disease management.

Six Multiplex TaqManTM-qPCR Assays for Quantitative Diagnostics of Pseudomonas Species Causative of Bacterial Blotch Diseases of Mushrooms.

Bacterial blotch is a group of economically important diseases of the common button mushroom (Agaricus bisporus). Once the pathogens are introduced to a farm, mesophilic growing conditions (that are optimum for mushroom production) result in severe and widespread secondary infections. Efficient, timely and quantitative detection of the pathogens is hence critical for the design of localized control strategies and prediction of disease risk. This study describes the development of real-time TaqManTM assays that allow molecular diagnosis of three currently prevalent bacterial blotch pathogens: "Pseudomonas gingeri," Pseudomonas tolaasii and (as yet uncharacterized) Pseudomonas strains (belonging to Pseudomonas salomonii and Pseudomonas edaphica). For each pathogen, assays targeting specific DNA markers on two different loci, were developed for primary detection and secondary verification. All six developed assays showed high diagnostic specificity and sensitivity when tested against a panel of 63 Pseudomonas strains and 40 other plant pathogenic bacteria. The assays demonstrated good analytical performance indicated by linearity across calibration curve (>0.95), amplification efficiency (>90%) and magnitude of amplification signal (>2.1). The limits of detection were optimized for efficient quantification in bacterial cultures, symptomatic tissue, infected casing soil and water samples from mushroom farms. Each target assay was multiplexed with two additional assays. Xanthomonas campestris was detected as an extraction control, to account for loss of DNA during sample processing. And the total Pseudomonas population was detected, to quantify the proportion of pathogenic to beneficial Pseudomonas in the soil. This ratio is speculated to be an indicator for blotch outbreaks. The multiplexed assays were successfully validated and applied by routine testing of diseased mushrooms, peat sources, casing soils, and water from commercial production units.

Molecular Epidemiology of Ralstonia solanacearum Species Complex Strains Causing Bacterial Wilt of Potato in Uganda.

Bacterial wilt (BW) caused by the Ralstonia solanacearum species complex (RSSC) is a serious threat to potato production in Uganda. However, little is known about the extent of the disease and the type of the pathogen strains involved. A nationwide survey was conducted to study BW prevalence and incidence in potato, and potato tuber and stem samples of potential alternative hosts were collected for pathogen isolation. DNA was extracted from pure cultures for genetic diversity studies. The pathogen was phylotyped by multiplex PCR; then, a subset of isolates was typed at sequevar level. Isolates of the same sequevar were then haplotyped using multilocus tandem repeat sequence typing (TRST) schemes. BW prevalence and incidence in potato farms were 81.4 and 1.7%, respectively. Three RSSC phylotypes were identified, with the majority of the strains belonging to Phylotype II (80%) followed by Phylotype I (18.5%) and III (1.5%). Phylotype I strains belonged to Sequevar 31, and Phylotype II strains belonged to Sequevar 1. Potato-associated Phylotype II Sequevar 1 strains were more diverse (27 TRST haplotypes) than nonpotato Phylotype I (5 TRST haplotypes). Mapping of TRST haplotypes revealed that three TRST haplotypes of Phylotype II Sequevar 1 strains play an important epidemiological role in BW of potato in Uganda being disseminated via latently infected seed.[Formula: see text]Copyright © 2019 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Detection of the Bacterial Potato Pathogens Pectobacterium and Dickeya spp. Using Conventional and Real-Time PCR.

Blackleg and soft rot of potato, caused by Pectobacterium and Dickeya spp., are major production constraints in many potato-growing regions of the world. Despite advances in our understanding of the causative organisms, disease epidemiology, and control, blackleg remains the principal cause of down-grading and rejection of potato seed in classification schemes across Northern Europe and many other parts of the world. Although symptom recognition is relatively straightforward and is applied universally in seed classification schemes, attributing disease to a specific organism is problematic and can only be achieved through the use of diagnostics. Similarly as disease spread is largely through the movement of asymptomatically infected seed tubers and, possibly in the case of Dickeya spp., irrigation waters, accurate and sensitive diagnostics are a prerequisite for detection. This chapter describes the diagnostic pathway that can be applied to identify the principal potato pathogens within the genera Pectobacterium and Dickeya.

Dickeya solani sp. nov., a pectinolytic plant-pathogenic bacterium isolated from potato (Solanum tuberosum).

Pectinolytic bacteria have been recently isolated from diseased potato plants exhibiting blackleg and slow wilt symptoms found in a number of European countries and Israel. These Gram-reaction-negative, motile, rods were identified as belonging to the genus Dickeya, previously the Pectobacterium chrysanthemi complex (Erwinia chrysanthemi), on the basis of production of a PCR product with the pelADE primers, 16S rRNA gene sequence analysis, fatty acid methyl esterase analysis, the production of phosphatases and the ability to produce indole and acids from α-methylglucoside. Differential physiological assays used previously to differentiate between strains of E. chrysanthemi, showed that these isolates belonged to biovar 3. Eight of the isolates, seven from potato and one from hyacinth, were analysed together with 21 reference strains representing all currently recognized taxa within the genus Dickeya. The novel isolates formed a distinct genetic clade in multilocus sequence analysis (MLSA) using concatenated sequences of the intergenic spacer (IGS), as well as dnaX, recA, dnaN, fusA, gapA, purA, rplB, rpoS and gyrA. Characterization by whole-cell MALDI-TOF mass spectrometry, pulsed field gel electrophoresis after digestion of whole-genome DNA with rare-cutting restriction enzymes, average nucleotide identity analysis and DNA-DNA hybridization studies, showed that although related to Dickeya dadantii, these isolates represent a novel species within the genus Dickeya, for which the name Dickeya solani sp. nov. (type strain IPO 2222(T) = LMG25993(T) = NCPPB4479(T)) is proposed.

Draft genome sequences of 17 isolates of the plant pathogenic bacterium dickeya.

Dickeya (formerly Erwinia chrysanthemi) species cause diseases on a wide range of crops and ornamental plants worldwide. Here we present the draft sequences of 17 Dickeya isolates spanning four Dickeya species, including five isolates that are currently unassigned to a species.

New Diversity of Ralstonia solanacearum Strains Associated with Vegetable and Ornamental Crops in Florida.

In 2003 and 2004, 15 isolates of Ralstonia solanacearum were obtained from wilting plants of field-grown pepper (Capsicum annuum) in south Florida and from pot-grown hydrangea (Hydrangea paniculata and H. macrophylla) and geranium (Pelargonium × hortorum) in commercial nurseries and retention ponds in north Florida. Diagnostic immunoassays and polymerase chain reaction (PCR) analyses identified all the isolates as R. solanacearum but not race 3 biovar 2. Pathogenicity studies on tomato, pepper, and tobacco revealed that all 15 strains had similar high virulence on tomato and all caused wilting of tobacco, although there were significant differences among the strains in aggressiveness on tobacco. An indigenous Florida tomato strain, race 1 biovar 1 (Rs5), caused no disease on tobacco and little or none on pepper. The three pepper strains were more aggressive than Rs5 or two hydrangea strains on all three pepper cultivars studied. Phylogenetic analysis based on an endoglucanase gene sequence indicated that these strains had three distinct origins. The three pepper strains belonged to phylotype I biovar 3 and clustered with strains from diverse hosts in Asia belonging to sequevar 13. The six geranium strains and four of the hydrangea strains were closely related to strains in sequevar 5, a distinct subcluster of phylotype II biovar 1 strains isolated from the French West Indies and Brazil. Two other biovar 1 strains from hydrangea and strains K60, AW, and Rs5 belonged to sequevar 7 in phylotype II and probably are native to North America. None of the Florida isolates belong to the highly regulated Select Agent race 3 biovar 2 subgroup, according to both the DNA sequence analysis and the biovar phenotypic test results. However, the race 3 biovar 2-specific B2 primers weakly amplified a product from some race 1 biovar 1 strains in real-time PCR, indicating that this assay may give false positives under some conditions. Given the high cost of a misdiagnosis, it seems advisable to use at least two independent diagnostic methods to confirm that a suspect isolate is R. solanacearum R3B2. This is the first report of the presence of R. solanacearum race 1 biovar 3 or phylotype I strains in North America, and the first report confirming R. solanacearum causing natural infection of hydrangea in Florida. Thus, R. solanacearum strains that are quite distinct from presumably indigenous strains are present and can infect diverse hosts in Florida.